Use of mCRP to enhance immune responses

ABSTRACT

The invention provides a method of enhancing an immune response to an immunogen in an animal. The method comprises administering to the animal an effective amount of the immunogen and an effective amount of a modified C-reactive protein (mCRP) or a mutant-mCRP. The invention also provides a vaccine and a method of producing this vaccine. The vaccine comprises an immunogen and an mCRP or a mutant-mCRP in a pharmaceutically-acceptable vehicle. The invention further provides a kit for immunizing an animal to an immunogen comprising (1) a container holding the immunogen and a container holding an mCRP or a mutant-mCRP or (2) a container holding the immunogen and an mCRP or a mutant-mCRP. The invention also provides a method of elicing an immune response to a hapten in an animal. The method comprises administering to the animal an effective amount of the hapten in association with an an effective amount of an mCRP or a mutant-mCRP. The invention further provides a vaccine and a method of producing this vaccine. The vaccine comprises a hapten and an mCRP or a mutant-mCRP in a pharmaceutically-acceptable vehicle. Finally, the invention provides a kit for immunizing an animal to a hapten comprising a container holding the hapten and an mCRP or a mutant-mCRP.

This application is a divisional of U.S. application Ser. No.09/376,628, filed Aug. 18, 1999 and claims benefit of provisionalapplication No. 60/128,888, filed Apr. 12, 1999.

FIELD OF THE INVENTION

The invention is concerned with enhancing immune responses. Inparticular, it has been discovered that modified C-reactive protein(mCRP) and mutant-mCRP can be used to enhance immune responses toimmunogens and to elicit an immune response to haptens.

BACKGROUND OF THE INVENTION

During injury, invasion of pathogens, or other forms of tissue damage,higher vertebrates implement a cascade of biochemical, immune andinflammatory reactions collectively termed the acute phase response. Theinflammation results in an increase in blood flow and the delivery ofimportant factors to the affected site. These factors act to limitmicrobial growth, reduce tissue damage, and aid in the removal ofdamaged tissue. The acute phase response is a primitive, nonspecificmechanism which reacts quickly prior to the development of the specificprocesses of humoral and cellular immunity.

C-reactive protein (CRP) has long been recognized as an important acutephase response protein, and its concentration in serum may increase asmuch as 1,000-fold during the acute phase response. CRP is a pentamerconsisting of five identical subunits, each having a molecular weight ofabout 23,500. The pentameric form of CRP is sometimes referred to as“native CRP.”

In about 1983, another form of CRP was discovered which is referred toas “modified-CRP” or “mCRP.” The formation of mCRP from native CRPinvolves the dissociation of native CRP into its subunits which alsoundergo a change in conformation. As a result, mCRP expressesantigenicity which is distinct from that of native CRP (referred to as“neo-CRP antigenicity”), and antibodies are available which candistinguish mCRP from native CRP (see, e.g., U.S. Pat. No. 5,272,258 andPotempa et al., Mol. Immunol., 24, 531-541 (1987)). The conversion ofnative CRP into mCRP is irreversible (the subunits do not reassembleinto native CRP). Kresl et al., Int'l J. Biochem. Cell Biol., 30,1415-1426 (1998).

It has been reported that mCRP can influence the development of monocytecytotoxicity, improve the accessory cell function of monocytes,potentiate aggregated IgG-induced phagocytic cell oxidative metabolism,and increase the production of interleukin-1, prostaglandin E andlipoxygenase products by monocytes. Potempa et al., Protides Biol.Fluids, 34, 287-290 (1987); Potempa et al., Inflammation, 12, 391-405(1988); Potempa et al., Proc. Amer. Acad. Cancer Res., 28, 344a (1987);Chu et al., Proc. Amer. Acad. Cancer Res., 28, 344a (1987); Zeller etal., Fed. Proc., 46, 1033a (1987); Chu et al., Proc. Amer. Acad. CancerRes., 29, 371a (1988). It is also known that mCRP can be used to treatviral infections, bacterial infections, endotoxic shock and cancer. SeeU.S. Pat. Nos. 5,283,238, 5,405,832, 5,474,904, and 5,585,349. It isfurther known that mCRP stimulates thrombocytopoiesis and the maturationof megakaryocytes and that it can be used to treat thrombocytopenia. SeeU.S. Pat. No. 5,547,931. Finally, it is known that mCRP binds immunecomplexes and aggregated immunoglobulin and can, therefore, be used toremove immune complexes and aggregated immunoglobulin from fluids and toquantitate immune complexes. See U.S. Pat. No. 5,593,897. It should benoted that mCRP differs from native CRP in its biological activities.See, e.g., the patents listed above.

SUMMARY OF THE INVENTION

The invention provides a method of enhancing an immune response to animmunogen in an animal. The method comprises administering to the animalan effective amount of the immunogen and an effective amount of amodified C-reactive protein (mCRP) or a mutant-mCRP, as further definedbelow.

The invention further provides a vaccine comprising an immunogen and anmCRP or a mutant-mCRP in a pharmaceutically-acceptable vehicle. Theinvention also provides a method of producing this vaccine. The methodcomprises combining the immunogen and an mCRP or a mutant-mCRP.

The invention further provides a kit for immunizing an animal to animmunogen. The kit may comprise a container holding the immunogen and acontainer holding an mCRP or a mutant-mCRP. Alternatively, the kit maycomprise one container holding both the immunogen and the mCRP ormutant-mCRP.

In addition, the invention provides a method of eliciting an immuneresponse to a hapten in an animal. The method comprises administering tothe animal an effective amount of the hapten in association with aneffective amount of an mCRP or a mutant-mCRP.

The invention further provides a vaccine comprising a hapten and an mCRPor a mutant-mCRP in a pharmaceutically-acceptable vehicle. The inventionalso provides a method of producing this vaccine. The method comprisescombining the hapten and an mCRP or a mutant-mCRP.

Finally, the invention provides a kit for immunizing an animal to ahapten. The kit comprises a container holding the hapten and the mCRP orthe mutant-mCRP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Graph of absorbance at 405 nm versus study day for miceimmunized with human lung adenocarcinoma A-549 peptides with amutant-mCRP (treated group) or with saline (no mutant—mCRP; controlgroup). Open circles—control group; closed circles—mutant-mCRP-treatedgroup.

FIG. 1B: Graph of absorbance at 405 nm versus study day for miceimmunized with human lung adenocarcinoma A-549 peptides with mutant-mCRP(treated group) or with saline (no mutant—mCRP; control group). Opencircles—control group; closed circles—mutant-mCRP-treated group.

FIG. 1C: Graph of % change in enzyme immunoassay (EIA) reactivity (Rx)versus study day for mice immunized with human lung adenocarcinoma A-549peptides with mutant-mCRP (treated group) or with saline (nomutant-mCRP; control group). Open circles—control group; closedcircles—mutant-mCRP-treated group.

FIG. 1D: Graph of % change from seven-day mean EIA Rx versus study dayfor mice immunized with human lung adenocarcinoma A-549 peptides withmutant-mCRP (treated group) or with saline (no mutant-mCRP; controlgroup). Open bars—control group; closed bars—mutant-mCRP-treated group.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS OF THEINVENTION

Modified-CRP can be prepared by using native CRP as the startingmaterial. The native CRP used for preparation of mCRP can be obtainedfrom natural sources (e.g., serum, plasma, pleural fluid or ascitesfluid). Methods of isolating native CRP from natural sources are knownin the art and are described, for example, by Volanakis et al., J.Immunol., 113:9-17 (1978); de Beer et al., J. Immunol. Meth., 50:17-31(1982); Potempa et al., Mol. Immunol., 24:531-541 (1987). CRP ispreferably isolated from serum, plasma, pleural fluid, or ascites fluidby calcium-dependent affinity chromatography usingphosphorylcholine-substituted BioGel® A 0.5 m (an agarose-based resinobtained from BioRad Laboratories, Richmond, Calif.). See, Potempa etal., Mol. Immunol., 24:531-541 (1987). Using this isolation method, CRPcan be obtained which is about 99% pure. Partially purified CRP may beobtained from commercial sources, such as Western States Plasma(Fallbrook, Calif.).

Native CRP can also be produced by recombinant DNA techniques. Genomicand cDNA clones coding for human, mouse, and rabbit CRP have beenisolated and sequenced. Tucci et al., J. Immunol., 131, 2416-2419(1983); Whitehead et al., Science, 221, 69-71 (1983); Lei et al., J.Biol. Chem., 260, 13377-83 (1985); Woo et al., J. Biol. Chem., 260,13384-88 (1985); Hu et al., Biochem., 25, 7834-39 (1986); Samols and Hu,Protides Biol. Fluids, 34, 263-66 (1986); Syin et al., J. Biol. Chem.,261, 5473-79 (1986); Ciliberto et al., Nucleic Acids Res., 15, 5895(1987); Hu et al., J. Biol. Chem., 263, 1500-1504 (1988); Whitehead etal., Biochem, J., 266, 283-90 (1990). Further, there is substantialhomology between the amino acid sequences of CRPs from differentspecies. For instance, there is from about 50% to about 80% sequencehomology between CRPs from various mammalian species. Hu et al.,Biochem., 25, 7834-39 (1986); Whitehead et al., Biochem, J., 266, 283-90(1990); and Kilpatrick et al., Immunol. Res., 10, 43-53 (1991). Giventhe substantial homology between CRPs from different species, probes canreadily be prepared from the known clones so that genomic and cDNAclones can be isolated which code for CRP from other species. Methods ofpreparing such probes and isolating genomic and cDNA clones are wellknown. See, e.g., Lei et al., J. Biol. Chem., 260, 13377-83 (1985); Wooet al., J. Biol. Chem., 260, 13384-88 (1985); Hu et al., Biochem., 25,7834-39 (1986); Hu et al., J. Biol. Chem., 263,1500-1504(1988);Whitehead et al., Biochem. J., 266,283-90(1990). To is obtain nativeCRP, eukaryotic host cells, preferably mammalian host cells, should beused for the expression of the CRP clone. See Samols and Hu, ProtidesBiol. Fluids, 34, 263-66 (1986); Hu et al., J. Biol. Chem., 263,1500-1504 (1988).

Methods of making mCRP from native CRP are known in the art (See, e.g.,Potempa et al., Mol. Immunol., 20, 1165-1175 (1983); Potempa et al.,Mol. Immunol., 24, 531-541 (1987)). For instance, mCRP can be preparedby denaturing CRP. CRP can be denatured by treatment with an effectiveamount of urea (preferably 8M) in the presence of a conventionalchelator (preferably ethylenediamine tetraacetic acid (EDTA) or citricacid). Further, CRP can be treated to produce mCRP by adjusting the pHof the protein to below about 3 or above about 11-12. Finally, mCRP canbe produced by heating CRP above 50° C., for a time sufficient to causedenaturation (preferably at 63° C. for 2 minutes), in the absence ofcalcium or in the presence of a chelator.

Monomeric preCRP, produced by cell-free translation of DNA coding forit, expresses neo-CRP antigenicity. preCRP is a precursor proteinconsisting of a signal or leader sequence attached to the N-terminus ofthe CRP subunit. During normal processing, the signal or leader sequenceis cleaved from the preCRP molecules to produce mature CRP subunitswhich assemble into pentameric native CRP. This normal processing andassembly occur in eukaryotic cells. See Tucci et al., J. Immunol., 131,2416-2419 (1983); Samols and Hu, Protides Biol. Fluids, 34, 263-66(1986); Hu et al., J. Biol. Chem., 263, 1500-1504 (1988). Therefore,mCRP can be prepared directly by recombinant DNA techniques by selectingconditions so that the CRP subunits are not assembled into pentamericnative CRP. This can be accomplished by expressing a desired genomic orcDNA clone in prokaryotic cells (referred to herein as“recombinant-mCRP” or “r_(m)CRP”). Recombinant-mCRP produced inprokaryotic cells consists of CRP subunits, preCRPs and/or fragments ofthe subunits and preCRPs. The CRP subunits and preCRPs may have slightlyaltered N-terminal and C-terminal sequences which reflect or assisttheir production in prokaryotic cells. For instance, they may havemethionine as the N-terminal amino acid.

Therefore, as used herein, the terms “modified-CRP” and “mCRP” meanpreCRPs or subunits of CRP, in free or aggregated form, which expressneo-CRP antigenicity. The terms comprise all of those forms of mCRPdescribed above, including CRP subunits and preCRPs having slightlyaltered N-terminal and C-terminal sequences which reflect or assisttheir production in prokaryotic cells. Neo-CRP antigenicity can bedetected using antibodies specific for mCRP (see, e.g., U.S. Pat. No.5,272,258 and Potempa et al., Mol. Immunol., 24, 531-541 (1987)) instandard immunoassays. Further, given the substantial homology betweenthe amino acid sequences of CRPs from different species, it is expectedthat mCRP from any species will be effective in the practice of theinvention.

To avoid the aggregation of the CRP subunits and preCRPs that generallyoccurs when DNA coding for preCRP is expressed in prokaryotic cells,mutant CRP subunits and preCRPs have been developed. These mutant CRPsubunits and preCRPs contain one or more amino acid changes that produceCRP subunits and preCRPs that are less likely to aggregate when producedin prokaryotic cells. The amino acid(s) added, deleted and/or replacedare also chosen so that the mutant protein retains the neo-CRPantigenicity characteristic of mCRP.

Suitable amino acid changes include the deletion or replacement of atleast one, preferably all, of the cysteines in an unmutated CRP subunitor unmutated preCRP. CRP subunits contain two cysteines and preCRP'scontain three cysteines, and it is believed that some of these cysteinesform intermolecular disulfide bonds, thereby contributing to theformation of non-dissociable cross-linked aggregates. Therefore, one ormore, preferably all, of these cysteines are desirably deleted orreplaced. When the cysteines are replaced with other amino acids, theyare preferably replaced with glycine, alanine, valine, leucine,isoleucine, serine, threonine or methionine, but any amino acid can beused. Most preferred is substitution with alanine. Lysine andderivatized lysine residues may also contribute to non-dissociablecross-linking. Accordingly, suitable amino acid changes may also includethe deletion or replacement of at least one of the lysines in anunmutated CRP subunit or unmutated preCRP. As a result of the amino acidchanges in them, the mutant proteins are easier to purify with muchhigher yields than unmutated CRP subunits or unmutated preCRP's.

Not all of the amino acid additions, deletions and replacements needcontribute to the reduced likelihood of forming non-dissociableaggregates as long as the combined effect of all the changes is areduction in intermolecular non-dissociable cross-linking. For instance,the recombinant DNA manipulations used to produce the mutant proteinsmay result in amino acids being added at the amino or carboxy terminalends of the CRP subunit. This is acceptable as long as these amino acidsdo not contribute to the production of nondissociable aggregates. Inaddition, some of the amino acid changes may be made for other purposes.For instance, it is desirable to make amino acid changes which increasethe solubility of the resultant mutant protein in aqueous media, since amore soluble mutant protein is easier to purify and process. Suitableamino acid changes to increase the solubility include deleting one ormore hydrophobic amino acids, replacing one or more hydrophobic aminoacids with charged amino acids, adding one or more charged amino acids,or combinations of these changes. However, for the reasons stated above,it may be desirable to avoid the addition of lysine residues. Aqueousmedia include water, saline, buffers, culture media, and body fluids. Asanother example, amino acid changes can be made for the purpose ofproviding for the association of an immunogen or a hapten with themutated CRP subunit or preCRP (see below).

The mutant proteins can be prepared by expression of DNA coding for themin transformed host cells. DNA coding for a mutant protein can beprepared by in vitro mutagenesis of a known or newly-isolated CRPgenomic or cDNA clone or can be chemically synthesized. In vitromutagenesis techniques are conventional and well known. Particularlypreferred is site-directed mutagenesis using polymerase chain reaction(PCR) amplification. See, e.g., U.S. Pat. No. 5,547,931. The followingreferences described other site-directed mutagenesis techniques whichcan be used to produce DNA coding for a mutant protein: CurrentProtocols In Molecular Biology, Chapter 8 (Ansubel ed. 1987); Smith &Gilliam, Genetic Engineering Principles And Methods, 3, 1-32 (1981);Zoller & Smith, Nucleic Acids Res., 10,6487-6500 (1982); Zoller et al.,Methods Enzymol, 100,468-500 (1983); Zoller & Smith, DNA, 3, 479-88(1984); Brake et al., Proc. Natl. Acad, Sci. USA, 81, 4642-46 (1984);Bio/Technology, pages 636-39 (July 1984); Botstein et al., Science, 229,1193 (1985); Kunkel et al., Methods. Enzymol., 154, 367-82 (1987).

DNA coding for a mutant protein of the invention can also be prepared bychemical synthesis. Methods of chemically synthesizing DNA having aspecific sequence are well-known in the art. Such procedures include thephosphoramidite method (see, e.g., Beaucage and Caruthers, TetrahedronLetters, 22, 1859 (1981); Matteucci and Caruthers, Tetrahedron Letters,21, 719 (1980); and Matteucci and Caruthers, J. Amer. Chem. Soc., 103,3185 (1981)) and the phosphotriester approach (see, e.g. Ito et al.,Nucleic Acids Res., 10, 1755-69 (1982)).

Therefore, as used herein, the term “mutant-mCRP” means preCRPs orsubunits of CRP having a sequence mutated as described above whichexpress neo-CRP antigenicity. As noted above, neo-CRP antigenicity canbe detected using specific antibodies in standard immunoassays. Further,given the substantial homology between the amino acid sequences of CRPsfrom different species, it is expected that mutant-mCRPs derived fromthe preCRPs or CRP subunits of any species will be effective in thepresently claimed invention.

For a detailed description of the physical and chemical properties,biological activities, and methods of making mCRP, including r_(m)CRP,and mutant-mCRP, and antibodies to neo-CRP antigenicity, see U.S. Pat.Nos. 5,272,258, 5,283,238, 5,405,832, 5,474,904, 5,547,931, 5,585,349,5,593,897, and 5,874,238, published PCT application WO 94/18999, andU.S. patent applications Ser. Nos. 08/480,270, 08/548,974, 08/549,013and 08/767,795, the complete disclosures of which are incorporatedherein by reference.

Fragments of CRP subunits and preCRPs, having a native or mutantsequence, may have the same activities described herein for mCRP andmutant-mCRP, and the use of such fragments is considered to come withinthe scope of the present invention. It is also believed that proteinssubstantially homologous to CRP will have the activities describedherein for mCRP, and such proteins are also considered to come withinthe scope of the present invention.

The present invention is based on the discovery that mCRP andmutant-mCRP can act as adjuvants. As used herein, the term “adjuvant”means a substance that non-specifically enhances the immune response toan immunogen. “Immune response” means the specific humoral orcell-mediated response to an immunogen. Thus, antibody production orcell-mediated immunity is more vigorous in an animal receiving mCRP ormutant-mCRP than it is when the immunogen is given without the adjuvant.The fact that an immune response is enhanced can be determined bycomparative testing (e.g., administering an immunogen with and withoutmCRP or mutant-mCRP under otherwise identical conditions and measuringthe immune response, e.g., measuring antibody titers). In addition, theimmune response may be modified qualitatively (e.g., antibodies ofdifferent immunoglobulin classes may be stimulated).

Thus, mCRP or mutant-mCRP can be used to enhance an immune response toan immunogen in any species of animal that is capable of an immuneresponse. Preferably, the animal is a mammal, such as a rabbit, goat,dog, cat, horse or human. The mCRP or mutant-mCRP used as the adjuvantin a particular species of animal may be from the same species or adifferent species of animal. For instance, when the animal to beimmunized is a goat, goat mCRP, human mCRP, or mutant-mCRP derived fromgoat or human preCRPs or CRP subunits could be used as the adjuvant.However, to avoid an immune reaction to the adjuvant itself, mCRP fromthe same species that is to be immunized should be used. For instance,when it is desired to elicit an immune response to an immunogen in agoat, goat mCRP should be used as the adjuvant.

As used herein, an “immunogen” is any substance which stimulates animmune response (humoral immunity or cell-mediated immunity) in ananimal. Immunogens are either antigens or haptens that have beenmodified to be immunogenic.

Antigens are substances that elicit a specific immune response whenintroduced into an animal. An antigen may contain one or more antigenicdeterminants or epitopes. Antigens include polysaccharides, lipids,lipopolysaccharides, proteins, glycoproteins, lipoproteins,nucleoproteins, peptides, oligonucleotides and nucleic acids. Specificimmunogens include immunoglobulins, coagulation factors, peptide andprotein hormones (e.g., insulin, gonadotropin, somatotropin),interleukins, interferons, other cytokines, peptides comprising atumor-specific epitope (i.e., an epitope found only on a tumor-specificprotein), peptides containing a sperm-specific epitope (i.e., an epitopefound only on a sperm-specific protein), DNA, RNA, cells (e.g., redblood cells), cell-surface molecules (e.g., CD antigens, integrins, cellreceptors), microorganisms (viruses, bacteria, molds, and fungi, whichmay be intact, lysed, ground, or otherwise fragmented), fragments,portions, components, or products of microorganisms, etc.

Haptens are substances that do not initiate an immune response whenintroduced by themselves into an animal. Generally, haptens are smallmolecules of molecular weight less than about 1 kD. Haptens includesmall organic molecules (e.g., digoxin, heroin, cocaine, morphine,mescaline, lysergic acid, tetrahydrocannabinol, cannabinol, steroids,pentamidine, biotin) and small peptides (2-4 amino acids). To beimmunogenic, haptens must be modified. This is generally accomplished bycoupling haptens to an immunogenic carrier.

Suitable carriers are compounds capable of stimulating an immuneresponse to haptens coupled to them in a host animal. Many such carriersare well-known. For instance, the carrier may be a high molecular weightcompound. Suitable high molecular weight compounds include proteins,polypeptides, carbohydrates, polysaccharides, lipopoly-saccharides,nucleic acids, and the like of sufficient size and immunogenicity.Preferred high molecular weight compounds are proteins and polypeptides.Suitable immunogenic carrier proteins and polypeptides will generallyhave molecular weights between 4,000 and 10,000,000, and preferablygreater than 15,000. Such suitable carriers include proteins such asalbumins (e.g., bovine serum albumin, ovalbumin, human serum albumin),immunoglobulins, thyroglobulins (e.g., bovine thyroglobulin),hemocyanins (e.g., Keyhole Limpet hemocyanin), toxins (e.g., diptheriatoxoid, tetanus toxoid) and polypeptides such as polylysine orpolyalaninelysine.

Methods of coupling haptens to carriers are well known. For instance,peptides may be coupled to a carrier with conjugating reagents such asglutaraldehyde, a water soluble carbodiimide such as1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (ECDI),N-N-carbonyldiimidazole, 1-hydroxybenzotriazole monohydrate,N-hydroxysuccinimide, 6-maleimidocaproyl-N-hydroxysuccinimide,n-trifluoroacetylimidazole cyanogen bromide,3-(2′-benzothiazolyl-dithio) propionate succinimide ester hydrazides oraffinity labeling methods. See also Pierce Handbook and General Catalog(1989) for a list of possible coupling agents. The number of peptidesattached to the high molecular weight carrier is called the “epitopicdensity.” The epitopic density can range from 1 to the number ofavailable coupling groups on the carrier molecule. The epitopic densityon a particular carrier will depend upon the molecular weight of thecarrier and the density and availability of coupling sites. Preferably,only high molecular weight carriers having an epitopic density of atleast 15 peptides per molecule are used in the practice of theinvention.

To couple a small organic molecule hapten to a carrier protein,cross-linking chemical reagents can be used. Peeters, et al., J.Immunol. Methods, 120, 133-143 (1989); Pauillac, et al., J. Immunol.Methods, 220, 105-114 (1998); Kitagawa, J. Biochem., 94, 1165-1172(1983); Bauminger and Wilchek, Methods Enzymol., 70, 151-159 (1980). Oneapproach utilizes water-soluble NTHS-ester maleimide heterbifunctionalcross-linking reagent sulfo-MBS (Pierce Chemical Co., Rockford, Ill.).Amino functions on the hapten are covalently attached to thecross-linking reagent to form an active intermediate. This activatedintermediate can be linked to a carrier molecule (such as ovalbumin)which has been modified with a group-specific modifying agent (such as2-iminothiolane, which introduces additional free sulfhydryl groups onthe carrier protein). This modification increases the density ofreactive sites on the carrier so that, when the activated haptenintermediate is mixed with the modified carrier, a covalent linkage ofhapten to carrier can occur at a high hapten-to-carrier molar ratio. Ahigh hapten-to-carrier molar ratio is preferred to elicit a strongimmune response to the immunogen. Other methods of coupling smallorganic haptens to carriers are known. See, e.g., Catalog And Handbook(1994-1995) and Products Catalog (1997), Pierce Chemical Co., Rockford,Ill.

Additional references concerning conventional high molecular weightimmunogenic carrier materials and techniques for coupling haptensthereto are: Erlanger, Methods In Enzymology, 70, 85-104 (1980); Makelaand Seppala, Handbook of Experimental Immunology (Blackwell 1986);Parker, Radioimmunoassay of Biologically Active Compounds (Prentice-Hall1976); Butler J. Immunol. Meth., 7, 1-24 (1974); Weinryb and Shroff,Drug. Metab. Rev., 10,271-83 (1979); Broughton and Strong, Clin. Chem.,22, 726-32 (1976); Playfair et al., Br. Med. Bull., 30, 24-31 (1974);U.S. Pat. Nos. 4,990,596 and 4,782,136.

In one embodiment of the invention, the immunogen comprises a peptidehaving a sequence which includes a B-cell epitope linked to a peptidehaving a sequence which includes a T-cell epitope. Methods ofidentifying B-cell epitopes of a protein are known. See O'Hern andGoldberg, in Techniques In Protein Chemistry IV, pages 481-490 (1993);O'Hern and Goldberg, Proceed. Intern. Symp. Control Rel. Bioact. Mater.,20, 394-395 (1993). Three criteria are essential for immunogenicity: asize greater than 10 amino acids; surface accessibility of the sequence;and hypervariability (degree of foreignness). See O'Hern and Goldberg,in Techniques In Protein Chemistry IV, pages 481-490 (1993); O'Hern andGoldberg, Proceed. Intern. Symp. Control Rel. Bioact. Mater., 20,394-395 (1993). Methods of identifying T-cell epitopes are also known.See, O'Hern and Goldberg, in Techniques In Protein Chemistry IV, pages481-490 (1993); O'Hern and Goldberg, Proceed. Intern. Symp. Control Rel.Bioact. Mater., 20, 394-395 (1993). The three criteria for selection ofa T-cell epitope are: a size of 8-12 amino acids; hypervariability; andone or more representations of the tetrapeptide motif previouslyreported to be associated with T-cell epitopes. O'Hern and Goldberg, inTechniques In Protein Chemistry IV, pages 481-490 (1993); O'Hern andGoldberg, Proceed. Intern. Symp. Control Rel. Bioact. Mater., 20,394-395 (1993). Most preferably, the peptide has a sequence comprisingthe sequence of a promiscuous T-cell epitope. A promiscuous T-cellepitope is a T-cell epitope that is recognized by individuals of severaldifferent major histocompatability (MHC) types. Promiscuous T-cellepitopes are known. See, Ho et al., Eur. J. Immunol., 20, 477-483(1990); Kaumaya, et al., J. Molec. Recog., 6, 81-94 (1993). Apeptidewhich has a sequence comprising the sequence of a T-cell epitope mayinclude other sequences linked to the N-terminal or C-terminal of theT-cell epitope. In particular, additional amino acids may be provided tolink the B-cell epitope to the T-cell epitope. These linking amino acidsshould form a four-residue β-turn, based on examination of 33 patternsin native proteins that code for αα corners. Efimov, FEBS Lett., 166, 33(1984); Kaumaya et al., Biochemistry, 29, 13-23 (1990). Peptidescomprising a B-cell epitope may be coupled to a peptide comprising aT-cell epitope in the same manner as described above for couplinghaptenic peptides to high molecular weight proteins and polypeptides toform the immunogen. However, such immunogens are preferably synthesizedas a single peptide. Methods of synthesizing peptides are well known inthe art. For instance, peptides can be synthesized by solid phasesynthesis techniques or by recombinant DNA techniques.

To enhance an immune response, an effective amount of an mCRP or amutant-mCRP is administered to an animal. The mCRP or mutant-mCRP may beadministered in association with the immunogen, or the immunogen and themCRP or mutant-mCRP may be administered separately. When the immunogenand the mCRP or mutant-mCRP are administered separately, they may beadministered at separate sites, by separate routes and/or at differenttimes. Preferably, the immunogen and the mCRP or mutant-mCRP areadministered at approximately the same time and, most preferably, areadministered in association with each other.

An immunogen may be associated with an mCRP or a mutant-mCRP in avariety of ways. For instance, the immunogen and the mCRP or mutant-mCRPcan simply be mixed together and administered simultaneously to theanimal.

In such mixtures, neutral immunogens will associate with mCRP as aresult of hydrophobic interactions to form complexes, if the immunogenand mCRP are held in contact for a time sufficient (such times can bedetermined empirically) to allow the complexes to form. The contactingof the immunogen and the mCRP may take place in a solution of low ionicstrength or a solution of high ionic strength (see definitions below). Aneutral immunogen can be associated with a mutant-mCRP in this same way,provided that the amino acid changes in the mutant-mCRP do notsubstantially change the hydrophobicity of the mutant-mCRP as comparedto mCRP. Since mCRP has strong hydrophobic characteristics, neutralimmunogens can be complexed with most mutant-mCRPs in this manner.Anionic and cationic immunogens can be complexed as a result of ionicinteractions with an mCRP which has been altered to be more positivelyor negatively charged. Methods of derivatizing proteins to change theircharge are well known. See, e.g., Means and Feeney, ChemicalModification Of Proteins (Holden-Day Inc., San Francisco, Calif., 1971).For instance, positively-charged groups or negatively-charged groupscould be attached to the mCRP. Alternatively, the mCRP could bechemically treated to change its charge (e.g., by changingpositively-charged residues to negatively-charged residues). In anotheralternative, mutant-mCRPs mutated to be more positively charged or morenegatively charged could be used. The use of complexes formed as aresult of hydrophobic or ionic interactions is preferred to the use of asimple mixture.

It is known that mCRP is soluble in solutions of low ionic strength andthat it aggregates in solutions of high ionic strength. “Solution of lowionic strength” means a solution containing ≦0.05M NaCl or a solution ofanother salt having an equivalent relative salt concentration. “Solutionof high ionic strength” means a solution containing >0.05 M NaCl or asolution of another salt having an equivalent relative saltconcentration, including physiological solutions (about 0.15 M NaCl or asolution having an equivalent salt concentration). Thus, an immunogencan be associated with an mCRP by providing the mCRP in a low ionicstrength solution, adding the immunogen to the solution, and increasingthe ionic strength of the solution so that the mCRP aggregates, trappingthe immunogen in the aggregates. Thus, the resulting mCRP aggregateswill include the immunogen as part of the aggregates. An immunogen canbe associated with a mutant-mCRP in this same manner, provided that theamino acid changes in the mutant-mCRP do not substantially change thesolubility of the mutant-mCRP as compared to mCRP. Since mCRP has stronghydrophobic characteristics, an immunogen can be associated with mostmutant-mCRPs in this manner. The formation of aggregates is the mostpreferred method of associating an mCRP or a mutant-mCRP with animmunogen.

An immunogen and an mCRP or a mutant-mCRP also can be associated byattaching them both to the surfaces of liposomes. Methods of makingliposomes and attaching materials to their surfaces are well known inthe art. See, e.g. U.S. Pat. Nos. 5,283,238 and 5,858,399 and referencescited therein.

Finally, an immunogen can be associated with an mCRP or a mutant-mCRP bycovalent attachment to the mCRP or mutant-mCRP. Methods of covalentlyattaching compounds to proteins are well known (see above discussion ofattaching haptens to carriers).

The mCRP or mutant-mCRP and the immunogen can be administered in anyconventional manner, including orally, intradermally, subcutaneously,intramuscularly, nasally, etc. The preferred route of administration forboth the immunogen and the mCRP or mutant-mCRP is subcutaneously.

Effective amounts (effective dosages and number of doses) of the mCRP ormutant-mCRP necessary to elicit an enhanced immune response can bedetermined empirically as is known in the art. It is understood by thoseskilled in the art that the dose of the mCRP or mutant-mCRP that must beadministered will vary depending on, for example, the animal that willreceive the mCRP or mutant-mCRP, the route(s) of administration, whetherthe mCRP or mutant-mCRP and the immunogen are given together orseparately, and the age and size of the animal. Generally, a dose ofabout 0.01 to about 10 mg of an mCRP or a mutant-mCRP per kilogram ofbody weight of the animal will be effective to enhance an immuneresponse to an immunogen. It is also understood that it likely will benecessary to give more than one dose of the mCRP or mutant-mCRP. Ifmultiple doses must be given to the animal, the interval between dosesis preferably about 21 days. Administration of the mCRP or mutant-mCRPshould be continued until an acceptable immune response is achieved.

Effective amounts (effective dosages and number of doses) of theimmunogens are known in the art or can also be determined empirically.It is understood by those skilled in the art that the dose of immunogenthat must be administered will vary depending on, for example, theanimal that will receive the immunogen, the route(s) of administration,whether the immunogen and the mCRP or mutant-mCRP are given together orseparately, and the age and size of the animal. It is also understoodthat it may be necessary to give more than one dose of the immunogen. Ifmultiple doses must be given to the animal, the interval between dosesis preferably about 21 days. Administration of the immunogen should becontinued until an acceptable immune response is achieved.

The invention also provides vaccines comprising an immunogen and an mCRPor a mutant-mCRP in a pharmaceutically-acceptable vehicle.Pharmaceutically-acceptable vehicles are well known in the art. Forinstance, the vehicle may simply be a liquid, such as saline, buffers oran oil. It could also be a biodegradable polymer, such aspoly(lactic/glycolic acid) polymer. Gupta et al., Dev. Biol. Stand., 92,63-78 (1998); Jones et al., Behring Inst. Mitt., 98, 220-228 (1997). Thevaccines can also be provided in lyophilized form and reconstituted witha liquid, such as water or saline, just prior to use. It will beapparent to those persons skilled in the art that certain vehicles maybe more preferable depending upon, for instance, the route ofadministration and the nature of the immunogen employed in the vaccine.

The invention further provides a kit. The kit is a packaged combinationof one or more containers holding reagents and other materials usefulfor immunizing animals. Suitable containers for the reagents includebottles, vials, test tubes, syringes, and other containers known in theart. The kit may comprise one container holding an mCRP or a mutant-mCRPand one container holding an immunogen. Alternatively, the kit maycomprise one container holding both an immunogen and the mCRP ormutant-mCRP. The kit may also contain other materials which are known inthe art and which may be desirable from a commercial and userstandpoint, such as diluents, empty syringes, gauze pads, disinfectantsolution, etc.

Surprisingly, by the present invention, an immune response to a haptencan be elicited simply by associating the hapten with an mCRP or amutant-mCRP. Thus, the need to couple a hapten to a immunogenic carriermay be eliminated. The haptens can be associated with the mCRP ormutant-mCRP in the ways described above for immunogens. When used inassociation with haptens, the mCRP or mutant-mCRP is preferably, butneed not be, immunogenic in the species of animal to be immunized withthe hapten in order to obtain increased stimulation of the immuneresponse due to the immunogenicity of the mCRP or mutant-mCRP. Thus, themCRP or mutant-mCRP is preferably from a species of animal differentthan the species to be immunized with the hapten. Effective dosages androutes of administration of the hapten and the mCRP or mutant-mCRP canbe determined empirically and depend on the criteria described above forimmunogens. The invention also provides vaccines comprising a hapten inassociation with an mCRP or a mutant-mCRP and kits comprising acontainer holding a hapten in association with an mCRP or a mutant-mCRP.

EXAMPLES Example 1

Use of mCRP to Enhance the Immune Response to Tetanus Toxoid

Native CRP was isolated from pleural or ascites fluid bycalcium-dependent affinity chromatography usingphosphorylcholine-substituted BioGel® A 0.5 m (an agarose-based resinobtained from BioRad Laboratories) as described by Volanakis et al. (inJ. Immunol., 113:9-17 (1978)) and modified by Potempa et al. (asdescribed in Mol. Immunol., 24:531-41 (1987)). Briefly, the pleural orascites fluid was passed over the phosphorylcholine-substituted column,and the CRP was allowed to bind. Then, the column was exhaustivelywashed with 75 mM Tris-HCl-buffered saline (pH 7.2) containing 2 mMCaCl₂ until the absorbance at 280 nm was less than 0.02. The CRP waseluted with 75 mM Tris, 7.5 mM citrate-buffered saline (pH 7.2). Thishigh concentration of Tris significantly reduces non-specificallyadsorbed proteins which often contaminate affinity-purified CRPpreparations. CRP-containing fractions were pooled, dilutedthree-to-five fold with deionized water, adsorbed to Q-Sepharose FastFlow® ion exchange resin (Pharmacia), and then eluted with a linear saltgradient from 0-1M NaCl in 10 mM Tris-HCl, pH 7.4. CRP-containingfractions were pooled and re-calcified to 2-5 mM CaCl₂ (by adding asuitable amount of a 1M solution) and applied to unsubstituted Biogel® A0.5 m column to remove residual serum amyloid P component (“SAP”). Then,the CRP was concentrated to 1 mg/ml using ultrafiltration (Amicon; PM30membrane) under 10-20 psi nitrogen. A CRP extinction coefficient (mg/ml)of 1.95 was used to determine concentration. Next, the concentrated CRPwas exhaustively dialyzed against 10 mM Tris-HCl-buffered saline, pH7.2, containing 2 mM CaCl₂. This preparation produced a single Mr 23,000band on SDS-PAGE electrophoresis and was more than 99% free of SAP, IgGand all other proteins tested for antigenically.

To make mCRP, purified native CRP, prepared as described above, at 1mg/ml was incubated in 8M ultra-pure urea in the presence of 10 mM EDTAfor one hour at 37° C. The urea was removed by dialysis into 10 mMsodium phosphate buffer (pH 7.4) or Tris-HCl buffer (pH 7.2) containing0.015M sodium chloride. The mCRP was sterile filtered through a 0.2micron filter (Gelman, Ann Arbor, Mich.).

Sterile-filtered mCRP, 600 μg/ml in 25 mM Tris-HCl buffer, pH 7.4,containing 0.015 M NaCl, was diluted to 12.5 μg/ml, 25 μg/ml, or 50μg/ml in saline (0.15 M NaCl), and 0.1 ml of each mCRP concentration, or0.1 ml of saline (control), was admixed with 0.1 ml tetanus toxoid at0.25 μg/ml in saline. The dilution of the mCRP with saline caused themajority of the mCRP to aggregate, trapping the tetanus toxoid in theaggregates, and forming a suspension which was used for immunization.

Ten female mice, 4-6 weeks old, were immunized subcutaneously (s.c.) onday 0 with either the tetanus toxoid-mCRP suspension or with tetanustoxoid in saline. The immunizations were repeated on day 21.

Blood samples were drawn on days 21, 28 and 35, and titers of IgGantibodies to tetanus toxoid were determined by a standard enzymeimmunoassay (EIA) using tetanus toxoid immobilized in the wells of amicrotiter plate and serial dilutions of the sera from the immunizedmice. Binding isotherms were generated, from which 50% binding titerswere calculated.

The results are presented in Table 1 below. As can be seen from Table 1,after 4 and 5 weeks, IgG antibodies to tetanus toxoid were increased inall mCRP-treated animals, compared to saline-treated animals. Inparticular, IgG antibodies were increased 4-6 fold in mCRP-treatedanimals, compared to saline-treated animals, for the animals receiving50 μg/ml mCRP. No side effects of administering mCRP were observed.

TABLE 1 EIA Anti-IgG To Tetanus Toxoid Modified-CRP Day blood was drawnSaline 12.5 μg/ml 25 μg/ml 50 μg/ml 21  350 —  275   350 28 3000 41004200 12,800 35 2100 3600 6000 11,200

Example 2

Use of Mutant-mCRP to Enhance the Immune Response to Tumor-specificPeptide Immunogens

This example demonstrates the adjuvant activity of a mutant-mCRP in micereceiving immunogenic peptides comprising epitopes of a tumor specificantigen (named Labyrinthin) found on human adenocarcinoma A549 tumorcells. Three peptides were made using standard solid phase synthesismethods. Two of the peptides were ten amino acids in length, and the thethird was six amino acids in length. These three peptides have sequencesfound in the protein region of the :antigen recognized by monoclonalantibody 44-3A6. Radosevich et al., Cancer Res., 45, 5808-5812 (1985). Asolution of the peptides in saline at 10 μg/ml was prepared.

The mutant-mCRP was prepare as described in U.S. Pat. No. 5,874,238, thecomplete disclosure of which is incorporated herein by reference, byculturing Escherichia coli BLR(DE3) transformed with plasmid pIT13 (seeExample 1, particularly sections F-H, of U.S. Pat. No. 5,874,238).Plasmid pIT13 comprises DNA coding for a mutant human CRP subunit whichhas the same sequence as an unmutated human CRP subunit, except that ithas a methionine added at the N-terminal and the two cysteines atpositions 36 and 97 have been replaced by alanines.

Groups of 5 mice were injected subcutaneously (s.c.) on day 1 with 100μl of the solution of the peptides in saline on the right hindquarterand with either 100 μl of the mutant-mCRP (200 μg) or 100 μl saline onthe opposite (left) hindquarter. Starting on day 7, and continuing onceper week for 10 weeks, the mice were given additional 100 μl s.c.injections of the mutant-mCRP or saline on the left hindquarter.

Sera were collected at various times over a 12-week period. The bloodwas microfuged, the serum removed, and 1 μl of saturated sodium azidewas added to each serum sample. The sera were stored at 4° C. untilused.

The sera were assayed by EIA for IgG immunoreactivity to a cell lysateof A549 human adenocarcinoma cells. A549 cells were obtained from theATCC and grown in RPMI-1640 (90%), fetal calf serum (10%), withsupplements, under standard conditions. A549 cells abundantly expressthe antigen recognized by the monoclonal antibody 44-3A6. Confluentflasks were harvested by scraping the cells, washing them three timeswith sterile saline, and adjusting the cells to 1×10⁶ cells/ml. Thecells were then sonicated (3 pulses for 15 seconds on ice). 50 μl ofthis lysate was put in each microtiter plate (Costar) for 30 minutes. 50μl of 1.0% EM grade glutaraldehyde was then added to each well for 30minutes. The fluid was then removed, and the well flooded with 1% RIAgrade bovine serum albumin, for 30 minutes. Plates were stored at 4° C.in BSA/saline with azide. The mouse sera were diluted 1:200 in saline(with azide), and 50 μl aliquots were incubated in plates of A549lysates for one hour. After washing the plates, a secondary antimouseantibody (alkaline phosphatase conjugated) was added and the plates wereincubated for 1-3 hours. Substrate was added after washing the plates.Readings were taken after 2 hours, at 405 nm.

The results are presented in FIGS. 1A-D. In FIGS. 1A-B, the data areplotted as mean±standard error of group reactivity (FIG. 1A and FIG. 1Bshow data from two different experiments). The data were compared bycalculating the mean change on test dates from the pre-study mean forthat group of animals (FIG. 1C). The data were also compared bycalculating the mean change on all test dates from that level ofreactivity noted on day 7 (one week after the initial immunization)(FIG. 1D). As can be seen in FIGS. 1A-D, the human mutant-mCRP enhancedthe mouse IgG immune response to human adenocarcinoma-specific peptideantigens. The enhanced anti-adenocarcinoma immune response increasedabove control after about 7-8 weeks, and persisted at the elevated levelup until at least week 12. After defining the initial titers to theadenocarcinoma antigens on day 7, mutant-mCRP booster injections (withno further injections of antigens) led to continued increases in themouse IgG response to adenocarcinoma-specific antigens. After 7-8 weeks,IgG titers against adenocarcinoma antigen showed steady increases in themutant-mCRP-treated experimental group.

The multiple subcutaneous injections of mutant-mCRP were safelytolerated with no noted side effects. However, on study days 14 and 21,days when the second and third mutant-mCRP booster injections weregiven, a mild darkening of the skin was observed in mutant-mCRP-treatedanimals at the spot where the peptides were initially injected. Nodarkening of the skin was observed in the saline-treated animals or atthe site of the mutant-mCRP injections. This darkening of the skinaround the primary immunization site may also be an indication of theenhanced immune response of the mutant-mCRP-treated mice to theimmunogens.

We claim:
 1. A method of enhancing an immune response to an immunogen inan animal comprising administering to the animal an effective amount ofthe immunogen and an effective amount of a modified C-reactive protein(mCRP) or a mutant-mCRP, wherein the mCRP or the mutant-mCRP and theimmunogen are administered separately.
 2. A method of enhancing animmune response to an immunogen in an animal comprising administering tothe animal an effective amount of the immunogen and an effective amountof a modified C-reactive protein (mCRP) or a mutant-mCRP, wherein theimmunogen comprises a peptide.
 3. A method of enhancing an immuneresponse to an immunogen in an animal comprising administering to theanimal an effective amount of the immunogen and an effective amount of amodified C-reactive protein (mCRP) or a mutant-mCRP, wherein theimmunogen comprises a peptide, and wherein the peptide comprises atumor-specific epitope.